M. tuberculosis (Mtb) is one of the leading causes of death worldwide and claims millions of lives annually. Approximately ~1.7 billion people worldwide are asymptomatically infected with the tubercle bacillus and constitute a major impediment to worldwide public health control measures. The completed genome sequence of Mtb H37Rv emphasizes our inadequacies in understanding mycobacterial disease and pathogenesis and has revealed the need for an approach to convert raw genome sequence data into functional information. We believe that a significant bottleneck exits in the characterization of Mtb function, primarily because of the accumulation of more complex and detailed outputs (e.g., mycobacterial genome sequences, DNA microarrays experiments, large-scale protein expression measurements, and gene deletions), rather than focusing on what create these outputs in the first place. Furthermore, it has become increasingly clear that virulence and drug resistance pathways are mediated by networks of interacting proteins. To address this problem, we developed a simple and rapid method termed, Mycobacterial Protein Fragment Complementation (M-PFC) that allows us to analyze protein-protein association in mycobacterial cells. In Specific Aim 1, we will thoroughly evaluate and optimize M-PFC and develop quantitative assays to measure the strength of interaction, determine how well M-PFC can detect protein-protein association in the mycobacterial membrane, use M-PFC to study the effect of redox agents on protein-protein association, construct Gateway cloning vectors that will enable investigators to rapidly clone and analyze their genes of interest. Finally, we will test our system in Mtb. In Specific Aim 2, we will test and validate our system by screening a Mtb library for proteins that interact with a known virulence factor. This application is innovative in that it describes the development of a novel experimental system not yet described in the mycobacterial field and has strong potential to lead to the identification of new drug targets, and virulence factors involved in the persistence and pathogenesis of Mtb. This effort is also innovative in the impact that it will have on functional genomics, structural biology, drug design and systems biology. We will make our reagents available to the wider scientific community. [unreadable] [unreadable] M. tuberculosis (Mtb) is one of the leading causes of death worldwide and claims millions of lives annually. The completed genome sequence of Mtb H37Rv emphasizes our inadequacies in understanding mycobacterial disease and pathogenesis and has revealed the need for an approach to convert raw genome sequence data into functional information. We have developed a novel method termed M-PFC to study protein-protien association in mycobacteria. This system should allow the identification of virulence pathways involved in persistence and pathogenesis, which will improve control of TB. [unreadable] [unreadable] [unreadable] [unreadable]